Bicyclo aldehyde and process for preparation

ABSTRACT

Stable biologically active thromboxane A 2  analogues having the formula: ##STR1## wherein R 1  is OR 3 , where R 3  represents hydrogen or a pharmaceutically acceptable cation or lower alkyl group; or 
     R 1  is NR 4  R 5  where R 4  and R 5  are the same or different substituents selected from the group consisting of hydrogen and lower alkyl group; and 
     R 2  is hydrogen or an -OH group. 
     The thromboxane analogues are potent thrombotic agents, useful in cardiovascular treatment.

This is a divisional of application Ser. No. 087,678, filed Oct. 24,1979, now U.S. Pat. No. 4,260,806.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to stable, biologically active analogues ofthromboxane A₂ useful as thrombotic agents.

2. Brief Description of the Prior Art

The prostaglandins were first discovered in the 1920's and have provensince then to be among the most ubiquitous pharmaceutically activecompounds ever tested. Their use and the use of analogues andderivatives thereof, has been suggested in as wide a range ofapplications as fertility control, induction of labor, regulation ofblood pressure, regulation of blood clotting, control of asthma,anticonvulsion, antidepressing action and many others. A new compoundhas recently been discovered (Nature 263, 663 (1976); Prostaglandins,vol. 12, 685 and 715 (1976); Chem. and Engineering News, Dec. 20, 1976)which belongs to the general family of prostaglandins. The compound hasbeen named prostacyclin and its structure has been proven by synthesis(Johnson, et al, Prostaglandins, 12, 915 (1976); Corey et al, J. Amer.Chem. Soc., 99, 2006 (1977)) to be that of formula I. (The numberingsystem for prostacyclins is given for reference): ##STR2## Its genericname is 6,9α-oxido-11α,15α-dihydroxyprosta (Z) 5, E (13)-dienoic acid.Prostacyclin is the most potent inhibitor of blood platelet aggregationof all the prostaglandins discovered to date. It has also been shownthat prostacyclin destroys platelet aggregates after they have formedand that it has, in addition, a powerful action as a dilator of bloodvessels. A second compound, which acts in an exactly opposite way toprostacyclin, has also recently been discovered by Hamberg and coworkers(Proc. Nat. Acad. of Sciences, USA, 72, 2994 (1975)). This metabolite,named thromboxane A₂ (TA₂) and shown in formula II below has potentthrombotic and smooth muscle constricting properties: ##STR3## Bothprostacyclin (I) and TA₂ (II) are derived from a common intermediatecalled endoperoxide, which in turn is synthesized from arachidonic acidby the enzyme cyclooxygenase. Prostacyclin is rapidly decomposed to6-ketoprostaglandin F₁α (6-keto PGF₁α) and TA₂ is rapidly decomposed bythromboxane B₂ (TB₂), less active final products in both cases. Bothprostacyclin and TA₂ have very short half-lives under physiologicalconditions; that of prostacyclin being about 2 minutes and that of TA₂only a mere 30 seconds at pH 7.4 and 37° C. The lability of TA₂ iscaused by the presence of a sensitive bicyclic acetal system. Therelationships between these metabolites, their precursors, products, andthe enzymatic systems catalyzing their formations and decompositions,are summarized in Scheme I:

    ______________________________________                                         Scheme I                                                                     ______________________________________                                         ##STR4##                                                                      ##STR5##                                                                     Action on                                                                     ______________________________________                                        Platelet aggregation:                                                                      Inhibits      Promotes                                           Smooth muscle:                                                                             Relaxes       Constricts                                         ______________________________________                                                      ##STR6##                                                                                    ##STR7##                                          ______________________________________                                    

It can be seen that prostacyclin (produced by vascular endothelium) andthromboxane A₂ (produced by platelets) have opposite physiologicaleffects and are very short lived. The balance between the levels ofprostacyclin and thromboxane A₂, appears to maintain a finely tunedequilibrium between blood platelet aggregation versus dissolution andarterial constriction versus dilation.

Other important physiological effects which are mediated by the oppositetransient actions of prostacyclin and TA₂ are the maintenance of thenormal integrity of vessel walls, limitation of thrombus formation,assistance in the formation of hemostatic plugs by diminishedprostacyclin formation, blood pressure regulation, control ofinflammation, prevention of gastric ulceration and other similareffects. The pharmacological use of these metabolites however, isseverely hindered by their short half-lives, especially so in the caseof TA₂. Externally provided TA₂ will fail to reach its target tissuesintact in high enough concentrations to cause any effects. Furthermore,the need to maintain the drug in a totally anhydrous condition alsoprevents its ready shipment, storage and testing for pharmacologicalapplications. Therefore, if an analogue or derivative of TA₂ can befound which is stable and shows biological effects on blood plateletsand arteries, such analogue would have wide applications in pharmacologyand the treatment of cardiovascular and related diseases. The use ofsuch a stable analogue of TA₂ can be used for patients withcardiovascular diseases, such as thrombosis, heart attack, orarteriosclerosis. It can be used in shock, such as hemorrhagic shock.

Although several stable bioactive prostacyclin analogues have beenprepared (see, e.g., Nicolaou et al, Angewandte Chemie, Int. Ed.(English) 17, 293 (1978) and references cited therein; also U.S. patentapplication Ser. No. 886,141, filed Mar. 13, 1978) there have been,prior to this invention, only few reports of stable TA₂ analogues withbiological activity.

Nicolaou et al, (Proceed. of Nat. Acad. Sci. USA, 76, 2566 (1979))describe pinane thromboxane PTA₂ (formula III): ##STR8## This compoundis a stable, biologically active thromboxane A₂ analogue which inhibitscoronary artery contraction at normal and high concentrations. Itfunctions as an antagonist of natural TA₂. This compound (PTA₂) as wellas a variety of derivatives and analogues thereof are disclosed incopending U.S. application Ser. No. 19,932, filed Mar. 12, 1979 by K. C.Nicolaou and R. Magolda. The synthesis of PTA₂ has also been disclosedby Ansell et al at the Fourth International Prostaglandin Meeting heldin Washington, D.C., May 1979 (Abstracts of the Meeting, page 5).However, PTA₂ is the only stable TA₂ analogue reported to date. A needtherefore continues to exist for other stable thromboxane A₂ analogueswhich show biological activity and are useful in cardiovascular therapy.

SUMMARY OF THE INVENTION

It is therefore an object of this invention to provide a thromboticagent.

Another object of the invention is to provide a compound withvasoconstricting properties.

Still another object of the invention is to provide a stablebiologically active analogue of thromboxane A₂.

A further object of the invention is to provide a stable biologicallyactive analogue of thromboxane A₂ wherein the labile cyclic acetallinkages have been replaced by carbon atoms. Yet a further object of theinvention is to provide a method for the synthesis of a stablebiologically active analogue of thromboxane A₂.

Briefly, these and other objects of the invention which will hereinafterbecome more readily apparent, have been achieved by providingpharmaceutically active, stable analogues of thromboxane A₂ having theformula (IV) (together with the numbering used in this application):##STR9## wherein

R¹ is OR³, where R³ represents hydrogen or pharmaceutically acceptablecation or lower alkyl group; or

R¹ is NR⁴ R⁵, where R⁴ and R⁵ are the same or different and are selectedfrom the group consisting of hydrogen and lower alkyl group;

and wherein R² is hydrogen or --OH.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The compounds of the present invention are stable pharmaceuticallyactive thromboxane analogues of the formula (IV), shown below with anumbering system used in this application: ##STR10## wherein

R¹ is either OR³, where R³ represents hydrogen or a pharmaceuticallyacceptable cation or lower alkyl group; or

R¹ is NR⁴ R⁵, where R⁴ and R⁵ are the same or different and are selectedfrom the group consisting of hydrogen and lower alkyl group; and whereinR² is hydrogen or S- or R-hydroxy group.

Pharmaceutically acceptable cations useful for the purposes of thisinvention are for example pharmaceutically acceptable metal cations oramine cations.

Especially preferred metal cations are those derived from the alkalimetals, e.g., lithium, sodium and potassium, and from the alkaline earthmetals, e.g., magnesium and calcium, although cationic forms of othermetals, e.g., aluminum, zinc, and iron, are within the scope of thisinvention.

Pharmacologically acceptable amine cations are those derived fromprimary, secondary, or tertiary amines. Examples of suitable amines aremethylamine, dimethylamine, trimethylamine, ethylamine, dibutylamine,triisopropylamine, N-methylhexylamine, decylamine, dodecylamine,allylamine, crotylamine, cyclopentylamine, dicyclohexylamine,benzylamine, dibenzylamine, α-phenylethylamine, β-phenylethylamine,ethylenediamine, diethylenetriamine, and like aliphatic, cycloaliphatic,and araliphatic amines containing up to and including about 18 carbonatoms, as well as heterocyclic amines, e.g., piperidine, morpholine,pyrrolidine, piperazine, and lower-alkyl derivatives thereof, e.g.,1-methylpiperidine, 4-ethylmorpholine, 1-isopropylpyrrolidine,2-methylpyrrolidine, 1,4-dimethylpiperazine, 2-methylpiperidine, and thelike, as well as amines containing water-solubilizing or hydrophilicgroups, e.g., mono-, di-, and triethanolamine, ethyldiethanolamine,N-butylethanolamine, 2-amino-1-butanol, 2-amino-2-ethyl-1,3-propanediol, 2-amino-2-methyl-1-propanol,tris(hydroxymethyl)aminomethane, N-phenylethanolamine,N-(p-tertamylphenyl)diethanolamine, galactamine, N-methyl-glucamine,N-methylglucosamine, ephedrine, phenylephrine, epinephrine, procaine,and the like.

Examples of suitable pharmacologically acceptable quaternary ammoniumcations are tetramethylammonium, tetraethylammonium,benzyltrimethylammonium, phenyltriethylammonium, and the like.

Pharmaceutically acceptable lower alkyl groups are those derived from C₁-C₁₀ hydrocarbyl residues, especially C₁ -C₄. Most preferred are methyland ethyl groups.

When R² =OH, two possible isomers at position 16 are possible (16R and16S); the 16S isomer is preferred. The free acids(R¹ =OH), their esters(R¹ =Oalkyl), salts (R¹ =O-cation) and amides (R¹ =N(Alkyl)₂ or NH₂ orNH(alkyl)) of the thromboxane analogues are all encompassed by thepresent invention. Specific compounds of the present invention are forexample:

Compound IV wherein R¹ =R² =OH (R² is 16S).

Compound IV, wherein R¹ =OLi, ONa, OK or OCs and R² =OH (16S);

wherein R¹ =OCH₃ and R² =OH (16S);

wherein R¹ =N(CH₃)₂ and R² =OH (16S);

wherein R¹ =OH and R² =H;

wherein R¹ =OCH₃ and R² =H;

wherein R¹ =N(CH₃)₂ and R² =H.

Gorman, R. et al, PNAS, USA 74, 4007 (1977) and Fitzpatrick, F. A., etal, Nature, 275, 764 (1978) have shown that prostaglandin endoperoxide(PGH₂) analogues which lack an --OH group at the 15 position are stronginhibitors of platelet aggregation. Thus for example 15-deoxy-9,11-azo-PGH₂ - a compound which contains an azo functionality bridgingpositions, 9,11-, inhibits platelet aggregation while the 15 --OHanalogue has aggregating activity. 15-deoxy-9,11-epoxyimino-PGH₂,another stable PGH₂ analogue also inhibits platelet aggregation.Therefore, the thromboxane analogues of the present invention include inone of their preferred embodiments, compounds wherein R² =H, as shownabove for position 16.

The C₁ -amide derivatives of prostaglandin PGF₂, have been shown to beantagonistic to the action of natural PGF₂, free acids by Ramwell, P.and his coworkers (Ramwell, P. et al, Nature, 278, 549 (1978)). Te useof the C-1 amides in the present invention is thus also one of thepreferred embodiments.

The compounds of the present invention can be prepared from the bicyclicα,β unsaturated aldehyde V: ##STR11## by a series of condensation andalkylation reactions, which are essentially analogous to those describedin Nicolaou et al, Proc. Nat. Acad. of Sci., U.S.A., 76, 2566 (1979) andin Nicolaou and Magolda, co-pending U.S. application, Ser. No. 19,932,filed Mar. 12, 1979 and which is herein incorporated by reference. Theaforementioned references disclose the synthesis of pinane thromboxanePTA₂ (III) from aldehyde VIa: ##STR12## Aldehydes V and VIa differ inthat VIa bears gem-dimethyl groups at bridge carbon 6 while V does not.In the aforementioned reference, aldehyde VIa was easily obtained byoxidation from the naturally occuring pinane alcohol (-)-myrtenol(formula VIb). There is however no naturally occuring--and thus readilyavailable--bicyclic α,β unsaturated alcohol without a gem-dimethylfunctionality at carbon 6 which can be used in the present case. Thepresent inventors therefore had to develop a de novo complete synthesisof aldehyde VIa. In addition, to the best of Applicants' knowledge,aldehyde VIa has not been prepared before nor has a synthesis for itbeen suggested.

Aldehyde VIa can be prepared, according to this invention frombicyclo[3.1.1]heptan-2-one (formula VIIa): ##STR13## by treating VIIawith a reagent such as C₁ -C₄ alkoxy methyl triphenyl phosphorane ((C₁-C₄ alkyl)-O-CH=P(C₆ H₅)₃), preferably methoxy methyl triphenylphosphorane in a Wittig reaction, to yield the intermediate enol etherVIIb. The reaction is carried out in an excess of alkoxy methylphosphorane, preferably about 2:1 excess, at low temperatures such as-20° to +10° C., in an inert solvent. A mixture of geometric isomersVIIb is obtained at the double bond. The enol ether VIIb (as themixture) is then reacted with an aryl selenenyl halide such as phenylselenenyl chloride, or bromide to give the selenenide VIII: ##STR14##This reaction is carried out with a slight excess of the selenenylhalide reagent, preferably 1-1.5 equivalent excess, at very lowtemperatures, preferably -80° to 50° C. in an inert solvent such as ahalohydrocarbon. It yields after work up and purification, selenenideVIII which is then transformed into target aldehyde (V) by oxidationwith a 0-15% molar excess of an oxidation agent such as H₂ O₂,m-chloroperbenzoic acid or the like, at -80° to 50° C. in an inertsolvent, followed by treatment with base if needed to neutralize anyacid present in the medium. The yield of aldehyde V from selenenide VIIIis between 80 and 90% under these conditions.

The major problem of the synthesis is undoubtedly the construction ofthe strained bicyclo[3.1.1]heptane-2-one nucleus (VIIa). Although thisketone has been prepared in the prior art (Musso, H., et al, Chem. Ber.,100, 3614 (1967) and Gibson, T. J. Org. Chem., 37, 700 (1972)), theprevious approaches are synthetically unattractive and proceed in lowyields. The present inventors therefore investigated and developed twonovel approaches to strained bicyclic ketone VIIa.

In the first approach, bicyclic[2.1.1]hexan-2-one (formula IX below)(prepared according to Bond, F. T., et al, Org. Phot. Syn. 1, 31 (1971))was ring expanded by treatment with dibromomethane in base followed by acarbenoid-type rearrangement of the intermediate dibromoalcohol (X) withstrong base. The sequence of reactions is shown in the following scheme:##STR15## The preparation of dibromoalcohol X can be carried outaccording to Taguchi, H., et al, (J. Amer. Chem. Soc. 96, 3010 (1974))by the addition of dibromomethane (2.1-2.5 equivalents) to a cold (-80°to -50° C.) solution (in an ether such as THF, diethyl ether, or thelike) of IX in the presence of lithium diisopropyl amide (LDA)(1.5-2.5equivalent). Dibromoalcohol X can be obtained in up to 95% yield. Thering expansion of X to VIIa can be carried out according to Taguchi etal, J. Amer. Chem. Soc., 96 6510 (1974). Treatment of the dibromoalcoholwith an n-lower alkyl lithium, such as n-butyllithium, (2.0-2.5equivalent excess) in an ether (diethyl ether, THF or the like), at lowtemperatures (-50° to -80° C.) generates a β-oxidocarbenoid species XI:##STR16## which undergoes bond rearrangement to the desired ketone VIIa(when bond a migrates) contaminated with bicyclo[3.1.1]heptan-3-one, theregioisomer of VIIa (when bond b migrates). The ratio of desired ketoneVIIa to undesired regioisomer is about 6:1.

A second approach to ketone VIIa is more selective yet somewhat longerand involves the base-catalyzed cyclization of a ketone of formula XIIin dilute solution at -10° to +40° C: ##STR17## Y is a leaving groupcapable of being internally displaced by a base-generated α carbanion onsaid ketone of formula XII such as -acetyl, tosyl, trifluoroacetyl,trichloroacetyl, chloro, bromo, or the like. When an α-carbanion isgenerated in base on compound XII, the carbanion internally displacesthe leaving group Y (S_(Ni) reaction) yielding ketone VIIa. Preferably Yis tosyl and the internal displacement is carried out by 2.0-2.5equivalents of dimsyl potassium (KH-DMSO) preferably 2.1 equivalents, indilute DMSO solution at 15°-25° C. Ketone XII can be prepared from1.4-cyclohexadione (commercially available from Aldrich Chemical Co.) byfirst protecting one of the two ketone groups of 1.4-cyclohexadione withan acid-sensitive protecting group (PG) such as diethylene glycol, thencarrying out a Wittig-type condensation on the remaining, unprotectedketone group, with methyl triphenylphosphorane (CH₂ ═P(C₆ H₅)₃) understandard, well-known Wittig reaction conditions. This two-step sequenceyields compound XIII, wherein PG represents a standard ketone-protectinggroup (McOmie, Protecting groups in Organic Chemistry, PlenumPublishing, London, 1973). ##STR18## The olefinic functionality of XIIIis then hydrated at the primary carbon atom by the well-known borane/H₂O₂ reaction and, when Y is an acyl group, the resulting alcohol isfinally acylated (with an anhydride or acid chloride, such as tosylchloride or acetic anhydride for example) and its still protected ketoneis deprotected under appropriate, well-known conditions (McOmie, Supra).Final yields of ketone XII are about 75% based on1,4-dicyclohexanedione.

Having prepared the bicyclic α,β unsaturated aldehyde (V), all thatremains of the synthesis is to carry this aldehyde to the final productcarbocyclic thromboxane A₂ (formula IV). As mentioned supra, this can bedone by following the detailed instructions in Nicolaou et al, copendingU.S. application, Ser. No. 19,932, filed Mar. 12, 1979, which is hereinincorporated by reference. Briefly, aldehyde V is treated in aMichael-type 1,4-addition with an unsaturated anionic alkyl to givetrans-aldehyde XIV: ##STR19## Aldehyde XIV is then reacted at thealdehyde group in a Wittig reaction with an lower alkoxy-methylphosphorane followed by acid liberation of intermediate extendedaldehyde XV ##STR20## Finally extended aldehyde XV is reacted with acarboxybutyl phosphorane, in a Wittig reaction, to yield, afteresterification and/or amidation of the COO group and/or deprotection ofthe OH group, carbocyclic thromboxane IV. Preparation of the free acidor its corresponding salts can be carried by standard saponificationmethodology. When the 16-OH CTA₂ is prepared, two eprimers at C-16 areobtained.

The compounds of this invention can be administered by any appropriatemeans to warm-blooded animals. For example, administration can beparenterally, subcutaneously, intravenously, intramuscularly orintraperitoneally. Alternatively or concurrently, administration can beby the oral route. The dosage administered will be dependent upon theage, health and weight of the recipient, kind of concurrent treatment ifany, frequency of treatment, and the nature of the effect desired.Generally, daily dosage of active ingredient compounds will be fromabout 0.5 mg to 50 mg per kg of body weight. Normally, from 1 to 30 mgper kg per day, in one or more applications per day is effective toobtain the desired result. The compounds can be employed in dosage formssuch as tablets, capsules, powder packets, or liquid solutions,suspensions, or elixirs, for oral administration, or sterile liquid forformulations such as solutions or suspensions for parenteral use. Insuch compositions, the active ingredient will ordinarily always bepresent in an amount of at least 0.5% by weight based on the totalweight of the composition and not more than 90% by weight.

Unlike thromboxane A₂ (TA₂), the carbocyclic thromboxane A₂ (CTA₂) ofthe present invention is stable at ambient temperatures in solution orheat. CTA₂ exhibits potent vasoconstricting (cat coronary arteries)properties with potencies comparable to those of the natural thromboxaneA₂ yet possesses no intrinsic ability to agreggate platelets. CTA₂ alsoappears to be a potent inhibitor of arachidonic acid induced plateletaggregation. Nevertheless, CTA₂ induces sudden cardiac death in rabbitsprimarily by producing severe myocardial ischemia without coronarythrombosis.

Thus, in contrast to pinane thromboxane A₂ (PTA₂) which is a selectiveinhibitor of coronary artery constriction, platelet aggregation andthromboxane formation, the new analogue of this invention behaves partlyas a biological mimic to thromboxane A₂. This is a truly surprisingresult since the only structural differences between CTA₂ and PTA₂ arein the absence and presence of a gem-dimethyl functionality at carbonC-6 of the molecules, respectively. That such diametrically oppositebiological behavior would be obtained upon such relatively smallstructural change is quite unexpected. The thromboxane analogues of thepresent invention are useful in the treatment of thrombotic conditions,of blood clotting in heart attack cases, in artherosclerosis, diabetesand cerebral strokes. They are useful in the various types of shock,such as hemorrhagic shock.

Having generally described the invention, a more complete understandingcan be obtained by reference to certain examples, which are included forpurposes of illustration only and are not intended to be limiting unlessotherwise specified.

I. Experimental Methods of Biological Testing

Thromboxane A₂ analogues (up to 4 μl of a 2.5 mM solution in ethanol)were tested for their effects on cat coronary arteries continuouslyperfused with 10 ml Krebs-Henseleit solution as described below.

Cats of either sex (2.5-3.5 kg) were anesthetized with sodiumpentobarbital (30 mg/kg) given intravenously. Hearts were rapidlyexcised and placed in oxygenated (95% O₂ +5% CO₂) ice-coldKrebs-Henseleit (K-H) solution of the following millimolar composition:NaCl, 118; KCl, 4.75; CaCl₂ H₂ O, 2.54; KH₂ PO₄, 1.19; MgSO₄ 7H₂ O,1.19; NaHCO₃, 12.5; glucose, 10.00. A 20-gauge stainless steel cannulawas inserted into the right coronary artery via the coronary ostium.Distal to the cannula, approximately 1 cm of coronary artery wasdissected free of surrounding tissue. The section of right coronaryartery with the cannula in place was excised from the heart andimmediately transferred to a constant flow perfusion apparatus.

The perfusion apparatus consists of a reservoir containing 20 ml of warm(37° C.) oxygenated (95% O₂ +5% CO₂) K-H solution which bathes thecoronary artery and serves as recirculating perfusate. An increase inperfusion pressure signifies vasodilation. Following an initial 1 hr.equilibrium period, vascular responsiveness was established by adding 25mM KCl. After washing with fresh K-H solution for 20-30 minutes, thepreparation achieved a relatively constant low basal tone. Basalperfusion pressure averaged 50±2.5 mm Hg. Fresh K-H dilutions of stockthromboxane analog concentrations were added to the perfusate reservoirin 0.1-0.2 ml volumes. Changes in perfusion pressure in response tothromboxane analogue addition generally plateaus within 5 minutes ofadministration. Constriction of the arteries was induced by addition of15 or 30 nM 9,11-azo-prostaglandinendoperoxide (azo-PGH₂), or 1 μM9,11-methanoepoxy-PGH₂. These compounds and their effects are describedin the following references: Corey et al, PNAS, USA 72, 3355 (1975);Bundy, G. L., Tetr. Lett, 1957 (1975); Malmsten, C., Life Sci, 18, 169(1976) and Smith, J. B., et al, in Platelets and Thrombosis, Ed. byMills, D.C.B. et al, Academic Press, London/N.Y. 1977, pp 83-95.

Platelet aggregation was studied in an aggregometer (Chronolog Corp.,Phila., PA) using 0.5 ml citrated human platelet-rich plasma at 35° C.One minute after addition of analogue (up to 2 μl of a 25 mM solution inethanol) aggregation was initiated by addition of sodium arachidonate(0.3-0.5 mM), ADP (2 μM) collagen (1 μg/ml), epinephrine (50 μM),9,11-azo-PGH₂ (0.1-0.6 μM). The analogues were also tested for theireffects on the inhibition of ADP-induced aggregation by 2 nMprostacyclin or 20 nM prostaglandin D₂.

To study the effect of CTA₂ on intact animals, New Zealand white rabbits2-3.5 kg were anesthetized with pentobarbital sodium (25 mg/kg) andplaced in the supine position. The right femoral and external jugularveins and the left common carotid artery were cannulated withpolyethylene catheters. Standard limb needle electrodes were placedsubcutaneously for recording the electrocardiogram (ECG). The tracheawas cannulated with a glass connected to a pressure transducer for therecording of airway pressure. Mean arterial blood pressure (MABP),central venous pressure (CVP), airway pressure, and lead III of the ECGwere continuously recorded on a Grass Model 7 oscillographic recorder.Sodium arachidonate (95% pure, Sigma Chamical) at 2.0 mg/kg or CTA₂ (125μg/kg) were injected into the femoral arterial catheter, and the rabbitobserved until death. Two ml blood samples were drawn into 25 mM EDTAjust prior to injection of drug and just prior to death forradio-immunoassay of thromboxane B₂.

II. Chemical Results

1. Synthesis of Carbocyclic Thromboxane A₂ (CTA₂), Methyl Ester (IV) R¹=OH (16S and 16R configurations))

a. Preparation of Intermediate Bicyclo[3.1.1]heptan-2-one fromBicyclo[2.1.1]hexan-2one ##STR21## Bicyclo[2.1.1]hexan-2-one (Bond, F.T., et al, Org. Phot. Syn. 1, 31 (1971)) was ring expanded by a firstaddition of LDA (2.0 equiv) to a cold (-78° C.) solution of the bicyclohexan-2-one in the presence of 2.2 equiv. of dibromoethane, followed bytreatment of the resulting bromoalcohol intermediate with n-butyllithium(2.2 equiv) in ether at -78° C. to generate a 6:1 mixture ofbicyclo[3.1.1]heptan-2-one (desired isomer) andbicyclo[3.1.1]heptan-3-one (undesired regioisomers) in 75% total yield.The analytical data for this compound agreed with published data fromMusso, H, Chem. Ber., 100, 3614 (1967) and Gibson, T., J. Org. Chem. 37,700 (1972).

b. Preparation of Intermediate Bicyclo[3.1.1]heptan-2-one from1,4-cyclohexadione ##STR22## 1,4-cyclohexadione (Aldrich) was treatedwith excess ethyleneglycol (HOCH₂ CH₂ OH) under acidic conditions inrefluxing benzene followed by acetic acid/THF/H₂ O (3:2:2) treatment at45° C. for 15 hours to give the monoprotected compound. A Wittigreaction on the unprotected ketone group was then carried out with Ph₃P═CH₂ in DMSO at 25° C. to give 68% overall yield of the olefin. Thisolefin was then reacted with disiamyl borane/NaOH/H₂ O₂ (1:1:3) followedby tosylation of the resulting primary alcohol withtosylchloride/pyridine/-20° C. to give the protected tosylate, which wasdeprotected with AcOH-THF-H₂ O 2:1:1, 75° C., 2.5 hours, in 77% overallyield. The keto-tosylate was then cyclized in 65% yield tobicylco[3.1.1]heptan-2-one with dimsylpotassium (KH-DMSO) (2-1 equivs.)in dilute DMSO solution of 15°-25° C.

c. Conversion of Bicyclo[3.1.1]heptan-2-one to 2-carboxaldehyde,bicyclo[3.1.1]hept-2-ene ##STR23## Treatment ofbicyclo[3.1.1]heptane-2-one with methoxymethyl triphenylphosphorane (2equiv) in THF/toluene at 0° C. afforded an enol ether at the 2-positionas a mixture of E-and Z-isomers. The mixture was exposed to C₆ H₅ -Se-Cl(1.5 equivalents) in CH₂ Cl₂ at -78° C. to produce, after aqueouswork-up and chromatographic isolation an intermediate selenenide:##STR24## in 72% overall yield. Oxidation of the selenenide with m-CPBA(1.1 equiv) in CH₂ Cl₂ at -78° C. followed by addition of diisopropylamine (2.2 equiv) and warming to 25° C. resulted in the rapid formationof the desired title α,β-unsaturated aldehyde.

d. Preparation of Final Product from2-carboxaldehyde-bicyclo[3.1.1]-hept-2-ene

The lower chain of the thromboxane molecule was introduced by1,4-addition to the title α,β-unsaturated aldehyde, of the cupratereagent obtained from (+)-trans-lithio-1-octen-3-oltert-butyldimethylsilylether and 1-pentynylcopperhexamethylphosphoroustriamide complex (78% yield). The trans aldehyde(J_(HaHb) =10 Hz) was the major product of this reaction and wasobtained exclusively as the thermodynamically more stable isomer(mixture of 16-epimers) after exposure to potassium carbonate inabsolute methanol at 25° C. The upper side chain of the thromboxane wascompleted by (i) condensation of the trans-aldehyde withmethoxymethylenetriphenylphosphorane (1.5 equiv) in toluene-THF solutionat 0° C. furnishing an enol ether in 93% yield as a mixture ofgeometrical isomers (ii) quantitative liberation of the aldehyde fromthe enol ether by treatment with Hg(OAc)₂ -kl in aqueous THF at 25° C.and (iii) Wittig reaction of the resulting extended aldehyde with thesodium salt of 4-carboxybutyltriphenylphosphorane in DMSO obtained as amixture of diastereoisomers at the 16-position. After removal of thesilyl ether (AcOH-THF-H₂ O, 3:2:2, 45° C., 12h. 90 yield) the twodiastereoisomers Rf=0.25 and Rf=0.28 (ratio ca 1:1) were separatedchromatographically on silica gel plates using ethylacetate-petroleumether mixtures (7.5:92.5) as solvent. The isomer with Rf 0.25- assumedto be the 16 S isomer on the basis of chromatographic mobility andbiological activity, had the following analytical data:

¹ H NMR (360 MHz, CDCl₂)τ: 4.43-4.75 (m, 4H, olefinic) 5.93 (m, 1H,CHO), 6.33, (3H, s, COOCH₃), 7.62-8.80 (m, 26H), 9.01 (t, J=9 Hz, 1H),9.12 (t, J=5 Hz, 3H, CH₃); High resolution mass spec calc. for C₂₃ H₃₈O₃ : 362.2821 found: 362.2828; IR (CCl₄) νmax 3410 cm⁻¹ (OH), 1739 cm⁻¹(COOCH₃).

Note: It is possible to prepare the optically active 16S isomer directlyby starting with the optically active lithium/cuprate reagent, preparedform S-trans-lithio-1-octen-3-ol tert-butyldimethylsilyl ether and1-pentynylcopper hexamethylphophoroustriamide complex, instead of the(R,S-)-trans-lithio-1-octen-3-ol-tert butyldimethyl silyl ether.

2. Preparation of Carbocyclic Thromboxane A₂, free acid (IV, R¹ =OH, R²=--OH, 16S and 16R configurations)

Basic hydrolysis of the more polar compound obtained in Experiment 1(Rf=0.25) in THF/LiOH solution at 25° C. led quantitatively to the 16Scarbocyclic thromboxane A₂ analog whereas the less polar ester (Rf=0.28)after similar treatment afforded the 16R hydroxy epimer.

III. Biological Results

Carbocyclic thromboxane A₂ produced dose-dependent constriction (i.e.,increased perfusion pressure at constant flow) in isolated perfused catcoronary arteries. CTA₂ was found to be more potent a constrictor thanazo-PGH₂ >9,11-methanoepoxy-PGH₂ >9,11 epoxymethano-PGH₂>>PGH₂ >>Thromboxane B₂. CTA₂ was 10,000 times as potent as TB₂ withregard to the concentration required to produce an increase in coronaryperfusion pressure of 20 mm Hg (EC₂₀). CTA₂ was also 4 times as potentas 9,11-azo PGH₂ and 5-6 times as potent as the other two PGH₂derivatives (i.e., 9,11-methanoepoxy PGH₂ and 9,11-epoxymethano PGH₂).

CTA₂ was a thromboxane agonist at all concentrations, and did notantagonize the coronary constrictor effects of 9,11-methanoepoxy PGH₂even at concentrations up to 10⁻⁷ M. However, pinane thromboxane A₂, athromboxane antagonist, almost completely antagonized the coronaryconstrictor effects of CTA₂. Thus, CTA₂ behaved as a pure thromboxaneagonist on the coronary vasculature without any antagonist actions toendoperoxide-like analogs.

With regard to platelet aggregation, CTA₂ did not behave as athromboxane agonist. It failed to induce platelet aggregation in vitroup to concentrations of 100 μM. Moreover, CTA₂ was a very potentinhibitor of platelet aggregation to a variety of prostanoids includingarachidonic acid, 9-11-azo PGH ₂, 9,11-methanoepoxy PGH₂ and9,11-epoxymethano PGH₂. At 4 to 5 μM CTA₂ completely prevented inducedplatelet aggregation. However, lower concentrations (i.e., 1 to 4 μM)were only partially protective. In the case of arachidonic acid inducedaggregation, lower concentrations of CTA₂ (i.e., 1-2 μM) delayed theonset of platelet aggregation without actually inhibiting the magnitudeof the overall response, indicating an effect on the early phase ofplatelet aggregation.

The profile of CTA₂ appears to be unique amongst the thromboxane andendoperoxide analogs thus far reported. CTA₂ is a potent coronaryvasoconstrictor, which does not antagonize the constrictor effects ofendoperoxide analogs. It thus behaves as a potent thromboxane agonist onthe vasculature. However, CTA₂ acts as though it is a thromboxaneantagonist on platelet aggregation. It does not induce plateletaggregation itself, and it antagonizes the aggregatory action ofarachidonic acid and endoperoxide analogs. Moreover, it selectivelyinhibits the formation of thromboxane B₂.

It was therefore of considerable interest to assess the overall effectof CTA₂ in the intact animal to determine whether its coronaryvascoconstrictor effect or its inhibition of platelet activitypredominated.

The typical response of an anesthetized rabbit to 125 μg/kg CTA₂ givenintravenously is as follows: Within 1-2 min, arterial blood pressurestarted to decline, central venous pressure rapidly increased, andregistration rate increased markedly but the depth of respiration becamevery shallow. Additionally, the ECG indicated myocardial ischemia; bysix minutes, severe hypotension occurred, respiration became ineffectiveand ischemia became even more prominent; at nine minutes death ensued.Radioimmunoassay of blood samples from rabbits injected with 125 μg/kgCTA₂ revealed no increase in circulating TB₂ concentrations, valuesaveraging 2.2±0.7 pmoles/ml prior to CTA₂ injection and 2.3±0.7pmoles/ml just prior to death. On autopsy, no thrombosis or plateletaggregates could be detected in either the coronary or pulmonary bed,and this was verified by histological sections of the heart and lungs.

Having now fully described this invention it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionset forth herein.

What is claimed as new and intended to be covered by Letters Patentis:
 1. An intermediate useful for the synthesis of carbocyclicthromboxane, having the formula: ##STR25##
 2. A method of preparing theintermediate of claim 1 which comprises reactingbicyclo[2.1.1]hexan-2-one of formula (1) ##STR26## with 2.1-2.5equivalents of dibromomethane, thereby obtaining dibromoalcohol offormula (2): ##STR27## reacting dibromoalcohol (2) with 2.0-2.5equivalents of a n-lower alkyl lithium, thereby to obtainbicyclo[3.1.1]hept-2-one, of formula (3): ##STR28## reactingbicyclo[3.1.1]hept-2-one with about a 2:1 molar excess of a C₁ -C₄alkoxy methyl triphenylphosphorane to obtain an enol ether of formula(4): ##STR29## wherein R is C₁ -C₄ alkyl group; reacting said enol etherof formula (4) first with a 1.0-1.5 molar excess of an aryl selenenylhalide at -80° to -50° C., followed by oxidation with 0-15% excess of anagent selected from the group consisting of H₂ O₂ and m-chloroperbenzoicacid, to thereby obtain the aldehyde of claim 1.